Background: Cantharidin (CTD) extracted from the traditional Chinese medicine Mylabris has significant therapeutic effects on various tumors. However, the high toxicity of CTD can cause serious liver damage, although the related molecular mechanisms remain unclear.Methods: In this study, we established models of CTD-induced liver and L-O2 cell damage in mice in vivo and in vitro. Subsequently, liver function indicators were detected in mouse serum, while liver tissues were subjected to pathological and transmission electron microscopy observations. L-O2 cell activity was investigated using the CCK-8 assay, and the mRNA and protein expression of DNA damage-induced transcription factor 4 (DDIT4) in liver tissue and L-O2 cells was detected using qPCR, immunohistochemistry, and western blotting. Western blotting was also used to detect the expression levels of autophagy- and apoptosis-related proteins in liver tissue and L-O2 cells. After RNAi interference with DDIT4, Rap, and 3-MA treatment, autophagy and apoptosis of L-O2 cells were detected using western blotting, flow cytometry, transmission electron microscopy, and confocal microscopy.Results: Following CTD exposure, the mouse liver showed significant pathological damage and an increase in autophagic lysosomes, while the vitality of L-O2 cells showed a significant decrease. CTD led to a significant increase in the mRNA and protein levels of DDIT4 in both liver tissue and L-O2 cells, as well as a significant increase in LC3-II, Beclin1, and Bax, whereas p-mTOR and Bcl-2 were significantly decreased. Following DDIT4 interference and 3-MA treatment, the levels of autophagy and apoptosis induced by CTD in L-O2 cells were reduced. After Rap treatment, both autophagy and apoptosis of CTD-induced L-O2 cells were significantly enhanced.Conclusion: The molecular mechanism of CTD-induced toxicity in mouse liver and L-O2 cells is mainly through DDIT4/mTOR signaling pathway activation, leading to an increase in autophagy and apoptosis levels.

The aim of this study was to evaluate the nephrotoxicity and molecular mechanism of Evodiamine (EVO). We combined RNA sequencing (RNA-seq) and network toxicology (NT) screening of potential target genes and signaling pathways, used molecular docking to validate core targets, and detected the mRNA expression of the key genes through quantitative real-time polymerase chain reaction (qRT-PCR). After exposure to EVO, body weight of mice decreased significantly, and the levels of renal index, Blood Urea Nitrogen (BUN) and Creatinine (Cr) were significantly increased, with varying degrees of pathological damage to the kidneys. NT identified 125 intersecting targets of EVO exposure related to kidney injury, including AKT1, TNF, TP53, etc. Among the 2888 differentially expressed genes obtained from RNA-seq, 504 genes were up-regulated and 2384 genes were down-regulated. By integrating NT and RNA-seq, 24 intersecting targets were identified. Among them, TRPV1, NOS3, HSP90AA1, and PPARG were selected for molecular docking validation. The results indicated that EVO had the highest affinity for PPARG (−8.07 kcal/mol). The qRT-PCR results indicated that the expression of the Pparg and Hsp90aa1 genes was significantly down-regulated, and the expression of the Nos3 and Trpv1 genes was significantly up-regulated. Immunohistochemistry further confirmed that EVO inhibited the expression of HSP90AA1 and PPARG, while enhancing that of TRPV1 and NOS3. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis suggested that EVO-induced nephrotoxicity is related to signaling pathways such as inflammatory mediator regulation of TRP channels, the PPAR signaling pathway, and the Apelin signaling pathway. In summary, the nephrotoxic effect of EVO may be related to the inhibition of the PPARG signaling pathway, the activation of the TRPV1 channel, the reduction in HSP90AA1 expression, and the imbalance of the Apelin-NOS3 pathway. This study provides a theoretical reference for clarifying the potential mechanism of renal injury caused by EVO and guiding its safe use.